Liposomes can be used as a carrier for many drugs, especially for antitumor drugs (in particular chemotherapeutic drugs). Liposomes can reduce the distribution of drug in normal tissues, but increase the accumulation of drug in tumor tissues, thereby improving the therapeutic index of drug. The reason why a liposome can target passively to a tumor relates to the physiological properties of tumor tissue. Tumor blood vessels may have a pore size of up to 100-780 nm due to its rapid growth, while normal vascular endothelial cells have a typical space of about 2 nm. Therefore, liposomes can accumulate passively in tumor region if they can circulate for a relatively long period in blood and have a size of less than 200 nm, because after liposomes with small size are administered via intravenous injection, they can not enter normal tissues but can penetrate blood vessel of tumor region and arrive at treatment area.
However, it is not easy to achieve the therapeutic advantages of liposome, and the following four requirements have to be met: (1) the drug can be encapsulated in liposome in a good encapsulation efficiency and a sufficient drug loading; (2) the drug will not be released from the liposome during storage period in vitro; (3) there is not a notable drug leakage during blood circulation of liposomal drug; and (4) the drug can be released effectively and thereby exerting its therapeutic effects when liposomes are accumulated in the tumor region. With regard to the current liposome techniques, the former three problems have been solved well, therefore, the rational release in vivo of liposomal drug draws more attentions. One critical technical problem to be solved for developing some liposomal drugs is to effectively control the rational release of liposomal drugs after targeting to a tumor region. This is especially important for some drugs, such as mitoxantrone.
It was found by a liposome study group in Canada that a liposome formulation having a size of about 100 nm, which was prepared by using hydrogenated soybean phosphatidylcholine (HSPC) and cholesterol as phospholipid bilayer and loading drug by a 300 mM citric acid gradient, was not as good as free mitoxantrone. In order to improve the therapeutic effect of liposome, the group finally changed the composition of phospholipid bilayer into dimyristoyl phosphatidylcholine (DMPC) and cholesterol, and obtained a preparation with improved therapeutic indexes. However, the leakage of drug may increase during the storage period because the phase transition temperature of DMPC is about 21° C., so that the preparation may not be stable (Liposomal formulations of mitoxantrone, U.S. Pat. No. 5,858,397).
Neopharm Corporation of USA used another technique to develop a liposome formulation of mitoxantrone, in which a cardiolipin carrying negative charge was added to phospholipid bilayer. Due to the intensive interaction between cardiolipin and mitoxantrone, mitoxantrone could be inserted into the phospholipid bilayer in a passive loading mode. This passive loading technique is different from active loading technique. By virtue of active loading technique, a drug would deposit in the intraliposomal aqueous phase in a form of precipitation. The Phase I clinical study on the product of Neopharm indicated that liposome drugs could increase the possibility of occasional infection, compared with free drug. The development of this product was ceased in view of safety (Liposomal preparations of mitoxantrone, CN01817424.8).
Pacific Institute of Materia Medica (Changchou, China) also filed a patent application for a liposomal preparation of mitoxantrone (A liposomal injection of mitoxantrone or mitoxantrone hydrochloride and the process for making the same, CN200410041612.1). In this application, traditional pH value gradient method was used to load drugs. This application seeks to protect a formulation with a specific ratio, and does not disclose the effects of factors such as composition of phospholipids, kinds of buffer salts in internal aqueous phase, size of liposome, drug/liposome ratio, etc. on the therapeutic efficacy and toxicity of liposome.
Zhirong Zhang, et al of West China School of Pharmacy, Sichuan University also studied liposomal preparations of mitoxantrone. They used soybean phosphatidylcholine with a phase transition temperature of 0° C. (which is marketed under the trade name EPIKURON 200) to prepare liposomes of about 60 nm. In this article, only pharmacokinetics was studied without concerning toxicity and therapeutic efficacy of the obtained liposomal preparation. Relevant contents can be seen in “Preparation of long circulating mitoxantrone liposomes and its pharmacokinetics”, Zhirong Zhang, Botao Yu and Yisong Duan, Acta Pharmaceutica Sinica, 2002, Vol. 37, No. 6; Studies on preparation of long circulating mitoxantrone liposomes with transmembrane ammonium sulfate gradients, Zhirong Zhang, Botao Yu, Yisong Duan and Yuan Huang, Chinese Pharmaceutical Journal, 2002 Vol. 37, No. 12; and Study on the preparation techniques of mitoxantrone liposomes, Yisong Duan, West China Journal of Pharmaceutical Sciences, 2001 Vol. 16, No. 02.
In the above studies, the size of liposomes is usually controlled in the range of 80˜150 nm, since there is a consensus in the field of liposome that a liposome with a size of about 100 nm would have the best targeting efficiency (Pharmacol. Rev. 1999 51: 691-744.). However, as mentioned above, a liposome should not only have an excellent targeting efficiency, but also a sufficient release from liposome to exert its effect.
As indicated above, according to the prior field, the leakage of drug during blood circulation should be essentially avoid so that the drug could be effectively transferred to tumors, but this requirement also results in a difficulty of releasing the drug from the liposome when it is targeted to tumor region. In conventional processes for making liposomes, a drug is usually encapsulated by a active loading technique, in which the drug encapsulated in the liposome is present in a colloid precipitate form having no bioactivity, so that only when the drug is released effectively from the liposome, it can change into a therapeutic drug with bioactivity. If the release rate of drug is too slow, the drug can hardly exert its therapeutic actions even though it has been targeted effectively to the tumor region, and its therapeutic effect may be even inferior to an unencapsulated drug.
Therefore, there is an urgent need in the field for a liposomal preparation capable of delivering a drug with good targeting ability and releasing the drug in the targeted tissues effectively, and for a corresponding liposomal pharmaceutical preparation.